patogenesis ppp
TRANSCRIPT
-
7/30/2019 Patogenesis Ppp
1/56
Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1005
_____________________________ _____________________________
Pamoplantar Pustulosis.
Pathogenetic Studies with Special Referenceto the Role of Nicotine
BY
EVA HAGFORSEN
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001
-
7/30/2019 Patogenesis Ppp
2/56
Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) inDermatology and Venereology presented at Uppsala University in 2001
ABSTRACTHagforsen, E. 2001.Palmoplantar pustulosis. Pathogenetic studies with special
reference to the role of nicotine. Acta Universitatis Upsaliensis.ComprehensiveSummaries of Uppsala Dissertations from the Faculty of Medicine 1005. 56 pp.Uppsala. ISBN 91-554-4955-7.
Palmoplantar pustulosis (PPP) is a chronic disease of unknown pathogenesis. Most of the patients were smokers. High prevalence of a number of autoimmune diseases wasobserved among the patients (thyroid disease 14%, gluten intolerance 8%, diabetestype 1 3%).
Eosinophils and neutrophils were found in large numbers in the pustules. Massiveinfiltrates of lymphocytes and mast cells in the dermis below the pustule and anabnormal acrosyringial pattern indicate that the acrosyringium is the target for theinflammation. Immunofluorescence (IF) revealed decreased innervation of the sweatgland, outward migration of substance P-positive granulocytes in the acrosyringiumand an increased number of contacts between mast cells and nerve fibres in thedermis.
Distributions of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE)were studied, since they regulate the level of acetylcholine, the main inducer of sweating. The most intense AChE-like immunoreactivity (LI) was observed in theacrosyringium in the lowest part of the stratum corneum, corresponding to the site of the pustule in PPP. ChAT-LI in granulocytes and AChE-LI in mast cells weredemonstrated, which may have implications for inflammatory processes in general.
Nicotinic acetylcholine receptors (nAChR) are activated by acetylcholine but also by nicotine. Immunohistochemstry of -3 and-7 subtypes of the nAChRs showedthat the nAChR expression in healthy skin was influenced by smoking. A highlyabnormal -7 nAChR distribution in PPP skin was observed.
The levels of nAChR antibodies were elevated in 42% of the PPP sera, and 68% of these sera gave specific endothelial IF in the papillary dermis in skin from non-smokers. Positive IF in the acrosyringium was also noted in skin from smokers.
Conclusions:Smoking seems to induce up-regulation of an antigen in palmar skin.The results indicate that PPP is an autoimmune disease and that nicotine might have arole in the onset of the inflammation.
Key words:Palmoplantar pustulosis, smoking, sweat gland apparatus, neuropeptides,non-neuronal cholinergic system, nicotinic receptor antibodies, autoimmune disease.
Eva Hagforsen, Section of Dermatology and Venereology, Department of Medical Sciences, Uppsala University, University Hospital, SE-751 85 Uppsala, Sweden
Eva Hagforsen 2001
ISSN 0282-7476ISBN 91-554-4955-7
Printed in Sweden by Fyris-Tryck AB, Uppsala 2001
-
7/30/2019 Patogenesis Ppp
3/56
To
Jan-ke
and Johan, Martin, and Emma
Att vgar att frlora fotfstet fr en stund Att inte vgar att frlora sig sjlv
Sren Kierkegaard
-
7/30/2019 Patogenesis Ppp
4/56
-
7/30/2019 Patogenesis Ppp
5/56
5
CONTENTS
ABBREVIATIONS.. 7PAPERS INCLUDED.. 8INTRODUCTION.. 9
Palmoplantar pustulosis . 9 Inflammation 10
Autoimmunity . 12Normal histology of palmar and plantar skin ... 12
The eccrine sweat gland apparatus 13 Normal histology and function 13
Inflammation and the acrosyringium. 14Innervation.. 14
Interaction between nervous and immune systems .. 15The neuronal cholinergic system 15The non-neuronal cholinergic system 17
General ... 17The non-neuronal cholinergic system in the skin 18Effects of acetylcholine on cells.. 19
Nicotinic influence- on the neuronal chol inergi c system . 20- on the non-neur onal chol in ergic system . 20- on keratinocytes 21- on endothel ial cel ls ... 21
AIMS OF THE STUDY. 22
PATIENTS AND METHODS... 23Patients. 23
Paper I, II, III and IV.. 23 Anamnestic data.. 23
Clinical examination 23Blood samples.. 23Biopsies 23
Paper V 23Reference persons 24Immunohistochemistry 24
Peroxidase and alkaline phosphatase methods.. 24Immunofluorescence... 26Serum immunofluorescence 27
Western blot (paper III) 28Radioimmunoassay (paper V). 28Statistics 29
-
7/30/2019 Patogenesis Ppp
6/56
6
RESULTS AND DISCUSSION. 30Paper I ... 30
Anamnestic data. 30Clinical findings.. 32Inflammatory cells.. 32
The sweat gland and duct ... 33Paper II . 34 PGP 9.5, substance P and calcitonin gene-related peptide 34
Nerve fibres and their contacts with mast cells.. 35Neuropeptide immunoreactivity in granulocytes 36
Paper III 37 ChAT and AChE in the epidermis and sweat gland apparatus.. 37
ChAT and AChE in inflammatory cells... 39Paper IV 40
Nicotinic receptors in the epidermis and sweat gland apparatus.. 40Nicotinic receptors in inflammatory cells 42
Paper V.. 43 Serum antibodies to nicotinic receptors and the immunofluorescence pattern43
CONCLUSIONS. 45
ACKNOWLEDGEMENTS 46
REFERENCES 48
-
7/30/2019 Patogenesis Ppp
7/56
7
ABBREVIATIONS
ab antibody
ABC avidin-biotin complex
ACh acetylcholine
AChE acetylcholinesterase
APAAP alkaline phosphatase-anti-alkaline phosphatase
C5a complement factor 5a
CGRP calcitonin gene-related peptide
ChAT choline acetyltransferase
ECP eosinophil cationic protein
EPO eosinophil peroxidase
EPX/EDN eosinophil protein X / eosinophil derived neurotoxin
FcRI high affinity receptor for immunoglobulin E
FITC fluorescein isothiocyanate
HLA human leukocyte antigen
IF immunofluorescence
Ig immunoglobulin
IL interleukin
LI like immunoreactivity
mAChR muscarinic acetylcholine receptor MC mast cells
MHC major histocompatibility complex
MNL mononuclear leucocyte
mRNA messenger ribonucleic acid
nAChR nicotinic acetylcholine receptor
PAP peroxidase anti-peroxidase
PBS phosphate buffered saline
PGP 9.5 protein gene product 9.5PPP palmoplantar pustulosis
SP substance P
TRITC tetramethyl-rhodamine-isothiocyanate
VIP vasoactive intestinal peptide
-
7/30/2019 Patogenesis Ppp
8/56
8
PAPERS INCLUDED
I. Eriksson MO, Hagforsen E, Pihl-Lundin I, Michalsson G: Palmoplantar pustulosis-a clinical and immunohistochemical study. Br J Dermatol. 1998;138: 390-398.
II. Hagforsen E, Nordlind K, Michalsson G: Skin nerve fibres and their contactswith mast cells in patients with palmoplantar pustulosis. Arch Dermatol Res.2000; 292: 269-74.
III. Hagforsen E, Aronsson F, Einarsson A, Nordlind K, Michalsson G: Thedistribution of choline acetyltransferase- and acetylcholinesterase-likeimmunoreactivity in palmar skin from patients with palmoplantar pustulosis.Br J Dermatol. 2000; 142: 234-42.
IV. Hagforsen E, Edvinsson M, Nordlind K, Michalsson G: Expression of -3and -7 subunits of nicotinic acetylcholine receptors in the skin of patientswith palmoplantar pustulosis. (submitted)
V. Hagforsen E, Mustafa A, Lefvert AK, Nordlind K, Michalsson G:Antibodies against nicotinic receptors in serum from patients with palmoplantar pustulosis. (submitted)
Reprints were made with permission from the publishers.
Cover photograph: Immunofluorescence pattern with PPP sera on normal palmar skin from a non- smoking control. Note the pattern in the papillary dermis. A photograph of a palm from a PPP patient is inserted.
-
7/30/2019 Patogenesis Ppp
9/56
9
INTRODUCTIONPalmoplantar pustulosisPalmoplantar pustulosis (PPP) is a chronic skin disease with an unknown
pathogenesis. It may be a localised form of pustular psoriasis, and occasionally the
patients have psoriasis-like lesions, particularly on the forearms and legs, but the
relationship is controversial. PPP is characterised by sterile intra-epidermal pustules
and usually also erythematous, scaly skin on the palms and soles. It is more common
in women than in men and is also more common in smokers than in non-smokers
(Eriksson et al 1998). The age at onset is usually between 20 and 60 years, 40-60
being most common.
Associations between PPP and autoimmune diseases such as autoimmune thyroid
disease have been reported (Rosn 1988). However, in that study no improvement of the palms and soles occurred when the thyroid disease was treated. A diabetic pattern
has been found in 22% of Japanese PPP patients at oral glucose tests (Uehara 1983)
but the clinical relevance of these tests is difficult to evaluate, since abnormal glucose
tolerance tests are not uncommon in middle-aged and elderly persons. Furthermore,
symptoms resembling rheumatoid arthritis have been noted in 13% of PPP patients,
which may be compared with a figure of 2.7% in the general population (Enfors and
Molin 1971).It is known that the pustules in PPP contain neutrophil granulocytes, but why these
cells are so abundant here is still unknown. There is a report, however, of intercellular
expression of interleukin-8 (IL-8) in the epidermis (Anttila et al 1992) of PPP skin
and IL-8 is a chemoattractant for neutrophils (Baggiolini et al 1989). Anttila et al also
observed strong IL-8 immunoreactivity in the whole eccrine sweat gland apparatus in
palmar skin from both PPP patients and control subjects. IL-8 has also been found in
sweat, and both this protein and its mRNA have been detected in sweat glandepithelium in abdominal skin (Jones et al 1995), indicating that IL-8 is produced in
situ.
There is no curative treatment for PPP today. Usually the patients with mild
symptoms are treated with emollients. Topical steroids are used in patients with more
severe forms and the most severe cases are treated with systemic drugs (retinoids,
cyclosporin). All these drugs are potent anti-inflammatory agents, which are virtually
independent of the cause of inflammation.
-
7/30/2019 Patogenesis Ppp
10/56
10
Inflammation
There are two fundamentally different types of defence against infection and tissue
damage, namely the innate response and the adaptive response.
Neutrophils are called the first line of defence, since within minutes of tissue
damage or pathological invasion they adhere to the endothelium of vessel walls and
migrate into the involved tissue. These cells form part of the innate immune response.
Other cells involved in the innate response are monocytes and macrophages
(phagocytic cells), basophils, mast cells and eosinophils (which release inflammatory
mediators), and natural killer cells. Neutrophils and macrophages have receptors for
antibodies and complement, a fact which enhances phagocytosis of microorganisms
coated with immunoglobulin and/or complement. Phagocytes also remove the bodys
own dead or dying cells. The most important chemotactic factors for neutrophils are
C5a (derived from complement), bacterial products (such as N-formyl-methionyl-
leucyl-phenylalanine), leucotriene B4 (product of arachidonic acid metabolism) and
IL-8.
Eosinophils are mainly tissue cells, and are most abundant in the gastrointestinal
tract, skin and lungs. Eosinophils are involved in processes in, for example, allergy
and parasitic infections. They contain different granular structures, of which specific
granules are most numerous. The specific granules, in turn, contain eosinophil
cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived
neurotoxin (EDN)) and eosinophil peroxidase (EPO). Activated eosinophils probably
kill parasites mainly by releasing ECP, instead of by phagocytosis. Eosinophils may
also induce neurotoxic effects by secreting EPX, and EPO has antibacterial
properties. In addition, eosinophils secrete prostaglandins, leukotrienes and various
cytokines. Lipids, complement components, cytokines and chemokines are known
eosinophil chemoattractants. Eotaxin is an example of a more recently discoveredeosinophil chemoattractant (Garcia-Zepeda et al 1996). Increased amounts of ECP in
the serum or in tissues have been shown to reflect increased turnover and/or increased
tissue activity of the eosinophils.
Basophils and mast cells (MC) are important in atopic allergies. Allergen binding to
IgE bound to high-affinity IgE receptors (FcRI) cross-links the FcRI, leading to
secretion of inflammatory mediators such as histamine, prostaglandins and
leukotrienes. Mast cells migrate into tissues, where they mature. They seem to be
-
7/30/2019 Patogenesis Ppp
11/56
11
localised in organs that are potential ports of entry of foreign agents, such as the skin,
lungs and gut. Mast cells have been found in tissues of patients with allergic diseases,
but these cells are also linked to chronic inflammatory disorders with a hitherto
unknown pathogenesis, for example psoriasis. They are divided into subsets on the
basis of their content of neutral serine proteases (Irani et al 1986). One subset, MCTC,
contains tryptase, chymase, cathepsin G and carboxypeptidase, whereas the other
subset, MCT, contains only tryptase. MCTC is found predominantly in the skin and in
the bronchial, nasal and intestinal mucosa, whereas MCT is localised mainly in
mucosal surfaces (Irani et al 1986; Irani et al 1989).
B and T lymphocytes (helper and cytotoxic) are involved in the adaptive (cell-
mediated) response. When T cells develop (in the thymus), there is a positive
selection in which cells that are able to interact usefully with peptides presented by
major histocompatibility complex (MHC) molecules survive, and a negative selection
in which cells reactive to self-proteins undergo apoptosis. Only 1% of the immature
precursor cells develop to immunocompetent T cells and are passed into the
circulation. The antigen-presenting cell is another important cell, which displays the
antigen to the T-cell receptor on the surface of helper T lymphocytes. The antigen is
presented by MHC class II molecules on the surface of dendritic cells. There are three
main kinds of class II molecules, HLA-DR, -DP and -DQ. When the receptors of the
T cells bind to antigens, the antigen-specific T cells proliferate (clonal selection).
Cytotoxic T cells have an antigen-specific T cell receptor, which recognises antigens
bound to MHC class I molecules (HLA-A, -B or -C), which are expressed on virtually
all cells. Self-cells that have been altered or infected are recognised and destroyed by
cytotoxic T cells. Mature B cells (plasma cells) produce antibodies against specific
antigens presented to them by helper T cells. Five different classes of antibodies
(immunoglobulins) are produced. One of them, immunoglobulin A (IgA), has animportant role in the first defence of the mucosal surfaces.
Cytokines, small soluble proteins, play a central part in the communication between
cells in the immune system and are also important for growth and differentiation of
haematopoietic, epithelial and mesenchymal cells. They regulate cell function in pico-
to nano-molar concentrations through specific receptors. There are pro-inflammatory
cytokines, e.g. IL-1, tumour necrosis factor , IL-6 and granulocyte-macrophage
colony stimulating factor, inflammatory cytokines, e.g. IL-2, IL-4 and interferon-
-
7/30/2019 Patogenesis Ppp
12/56
12
gamma (IFN ) and cytokines that are anti-inflammatory, e.g. IL-10, transforming
growth factor and IL-1 receptor antagonist. All these cytokines are involved to
varying degrees in different types of skin inflammation, including autoimmune
reactions, and are thus believed to have a role in the pathogenesis of psoriasis andrelated skin diseases.
AutoimmunityA disease is defined as autoimmune if the tissue damage is shown to be caused by an
immune response to self-antigens. Autoimmune disease can be triggered by
autoreactive T cells or by autoantibodies. The tissue may be damaged as a result of a
direct attack on the cells bearing the antigen, of immune complex formation or of
local inflammation. Another type of autoimmunity occurs when autoantibodies bind
directly to cellular receptors, causing either excess activity (e.g. Graves disease) or
inhibition of receptor function (e.g. myasthenia gravis).
Autoimmunity can be triggered by a variety of mechanisms, in most of which
infectious agents are involved. As a result of a tissue injury, antigens that are not
normally present in the circulation may be exposed to the immune response and not
recognised as a self-antigen. Infectious agents may induce either T- or B-cell
responses that can cross-react with self-antigens (molecular mimicry). Superantigens
produced by bacteria or viruses bind directly to the MHC class II molecule and
induce polyclonal T-cell activation leading to an autoimmune process.
Many human autoimmune diseases show HLA-linked associations, as may be
expected, since the ability of T cells to respond to a particular antigen depends on the
MHC type. It also seems as if sex hormones are involved in the pathogenesis of
autoimmune diseases. For example, systemic lupus erythematosus is more common in
women.
Normal histology of palmar and plantar skinIt is not known why PPP is localised to the palms and soles. However, the skin in
these locations differs from that in other parts of the body. Glabrous (non-hairy) skin
is characterised by a thick epidermis divided into several well-marked layers. There
are no hair follicles in glabrous skin, nor are there any sebaceous glands. In the
dermis of glabrous skin there are encapsulated sense organs, whereas the sensory
-
7/30/2019 Patogenesis Ppp
13/56
13
nerve endings in hairy skin are sometimes free, or terminate in hair follicles, and
others have expanded tips. The density of the sweat glands on the palms and soles is
600 to 700/cm2, compared to only 64 glands/mm2 on the back. Furthermore, the
stratum corneum is much thicker on the palms and soles, as a result of which the
outermost part of the sweat duct (the acrosyringium) has a well-developed coil
structure there, which is not so apparent in other sites.
The eccrine sweat gland apparatus Normal histology and function
The eccrine sweat gland apparatus consists of a secretory coil and a duct (Fig.1). The
coiled part is made up of the secretory coil and the proximal duct. The distal duct is
straight and connects the coil with the epidermis. In the epidermis and the stratum
corneum the duct forms a spiral (the acrosyringium) leading up onto the skin surface.
There are two main types of sweating: thermoregulatory and emotional (mental)
sweating. Thermoregulatory sweating occurs especially on the upper part of the trunk
and the face, but also on the palms and soles. Emotional sweating is provoked by
anxiety or pain and is characteristically associated with the palms, but its underlying
mechanisms are not known. The nerves surrounding the sweat glands are sympathetic
post-ganglionic fibres, which consist of non-myelinated class C nerve fibres, but
-
7/30/2019 Patogenesis Ppp
14/56
14
acetylcholine (ACh) is the principal neurotransmitter, acting via muscarinic receptors.
However, adrenaline may also induce palmar eccrine sweating (Wolf and Maibach
1974). Nicotine has been found capable of inducing axon reflex sweating by
iontophoresis (on the foot and leg) and produced a stained area (iodine starch
method) similar to that after ACh iontophoresis (Riedl et al 1998).
Inflammation and the acrosyringium
The acrosyringial epithelium possesses specialised keratinocytes, which are immune-
competent. Accordingly, expression of MHC class II HLA-DR, which play a critical
role in cell-mediated immune responses, has been observed on acrosyringial
epithelium in normal human skin (Murphy et al 1983). In a study of the antigenic
profile of the acrosyringium in normal skin (from the abdomen), McGregor el al
(1991) found expression of HLA-DR, -DP and -DQ on the acrosyringial epithelium,
and on the keratinocytes surrounding the acrosyringium they observed CD 36
(monocyte/platelet-specific molecule). Furthermore, expression of CD 68
(monocyte/macrophage-specific molecule) was detected on the acrosyringial
epithelium, but not on dermal ducts or sweat glands. Immunohistochemically,
Reitamo et al (1990) demonstrated IL-1 throughout the eccrine sweat gland apparatus.
The distal part of the acrosyringial epithelium showed intense staining. Didierjean et
al (1990) reported the presence of IL-1 in sweat from both truncal and palmar-
plantar regions, whereas IL-1 was detectable only in sweat from palms and soles,
indicating a site-dependent difference in the secretion of the two IL-1 molecules.
Furthermore, the IL-1 concentrations were much higher in the sweat during jogging
and sauna bathing than during spontaneous sweating, which they suggested could be
due to a stress-induced increase in the production of IL-1 by sweat gland cells. IgA,
which forms a defence barrier against microbial antigens on mucosal surfaces, has been detected in sweat secreted onto the skin surface (Imayama et al 1995), indicating
involvement of this immunoglobulin in the local immune defence of the skin.
Innervation
Most nerve fibres in the skin are sensory, and most of them are unmyelinated C and
myelinated A fibres that end as free nerve endings. Sensory nerve fibres express
several neuropeptides, which are biologically active polypeptides, and most
-
7/30/2019 Patogenesis Ppp
15/56
15
neuropeptide-containing fibres are located around blood vessels, sweat glands and
hair follicles or are present as free nerve endings. Neuropeptides can induce
neurogenic inflammation. Substance P (SP), calcitonin gene-related peptide (CGRP)
and vasoactive intestinal polypeptide (VIP), among other peptides, have been
demonstrated in nerve fibres in human skin (Wallengren et al 1987). Substance P-
containing fibres are most densly located in the palms, soles and axillary skin (Eedy
1993).
Human sweat glands are entangled with nerve fibres. Along the sweat duct, from
the gland to the surface of the skin, one or two nerve fibres are oriented. The nerve
fibres
around the sweat gland apparatus are reported to express CGRP, VIP and sparsely SP
(Kennedy et al 1994).
Interaction between nervous and immune systemsThere have been many reports indicating that the nervous system interacts with the
cutaneous immune system to mediate local inflammation. For instance neuropeptides
might be involved in skin diseases such as psoriasis [CGRP (Artemi et al 1997), SP
(Al'Abadie et al 1995); (Naukkarinen et al 1996), VIP (Anand et al 1991)], atopic
dermatitis [CGRP (Pincelli et al 1990), VIP (Ostlere et al 1995; Pincelli et al 1991)]
and eczema [VIP (Anand et al 1991)].
Some neuropeptides are able to degranulate mast cells (for example SP and VIP;
(Ebertz et al 1987; Lowman et al 1988) and also to induce vasodilatation and may
stimulate chemotactic and phagocytic activity of neutrophils and stimulate IgA
production by B cells [reviewed in Ansel et al (1996)].
It has been shown that neutrophil granulocytes contain VIP (ODorisio et al 1980),
that mast cells in patients with atopic dermatitis contain SP (Toyoda et al 2000) andthat activated T cells also contain SP (De Giorgio et al 1998).
The neuronal cholinergic system
Acetylcholine is the neurotransmitter of the cholinergic system. The synthesis of ACh
from coenzyme A and choline is catalysed by choline acetyltransferase (ChAT).
Acetylcholinesterase (AChE) is the cholinergic enzyme that hydrolyses ACh to
acetate and choline.
-
7/30/2019 Patogenesis Ppp
16/56
16
Acetylcholine acts on cells via two different classes of receptors, nicotinic (nAChR)
and muscarinic acetylcholine receptors (mAChR). Receptors of both classes are
found in the central nervous system.
Nicotinic receptors are also present in autonomic ganglia and at neuromuscular
junctions, while muscarinic receptors are found on autonomic effector cells
innervated by post-ganglionic parasympathetic nerves, and in blood vessels, where
they modulate vasoconstriction and dilatation (Furchgott and Zawadzki 1980).
The nicotinic AChRs are ligand-gated ion channels that mediate influx of Na+ and
Ca2+ and efflux of K + and are formed by various combinations of transmembrane
, , , and glycoprotein subunits. Each nAChR consists of five such subunits,
different combinations of which determine the functional and pharmacological
characteristics of the receptor (Conti-Tronconi et al 1994). The 1, 1, , and
subunits have been found at the neuromuscular junction. Neuronal nAChR consists of
combinations of the -2 to -9 and -2 to -5 subunits. Nicotinic AChRs formed by the
-4 and-2 subunits are the major subtype in the brain (Whiting et al 1991). The-3
subunit generally forms nAChRs together with the-5, -2, and-4 subunits (Conti-
Tronconi et al 1994). The-7, -8 and -9 subunits can form functional nicotinic
receptor channels of their own. The ACh-binding sites are believed to reside
primarily on the subunits (Papke 1993). However, -5 subunits, which are closely
related to-3 subunits, are believed not to be capable of forming an ACh-binding site
(Wang et al 1996). Alpha-7 nAChRs, which have five subunits, thus have five
putative binding sites for ACh (Fig. 2). It has also been demonstrated that -7
nAChRs are highly permeable to Ca2+ (Bertrand et al 1993).
Fig. 2. Subunit arrangements of two nAChR types around the central cation channel. To the left, thesubtype with3 as binding site with two putative ACh-binding sites; to the right, subtypes (e.g.7,8 or 9) forming functional homomers with five putative ACh-binding sites.
-
7/30/2019 Patogenesis Ppp
17/56
17
The muscarinic AChRs are glycoproteins with seven-helical transmembrane
segments, and they are coupled to G proteins (Hulme 1990; Hulme et al 1990). There
are five known subtypes, m1 to m5. The muscarinic receptor subtypes m1, m3 and
m5 mediate, upon stimulation, an activation of phospholipase C activity, resulting inan increase in the Ca2+ concentration; m2 and m4 mediate, upon stimulation,
inhibition of adenyl cyclase activity, resulting in decreased cyclic AMP formation
(Hulme 1990; Hulme et al 1990).
Neuronal nAChRs may be involved in neuronal diseases. It has been suggested, for
example, that-7 nAChRs may be associated with some aspects of schizophrenia.
Freedman et al (1995) found a decrease in -7 nAChR in the hippocampus of brains
from schizophrenia patients, and a decreased level of the -7 nAChR subunit proteinhas also been observed in the frontal cortex of schizophrenic brain (Guan et al 1999).
A high proportion of schizophrenic patients are intensive tobacco users (Lohr and
Flynn 1992), and it has been proposed that they may be attempting to self-medicate
(Dalack et al 1998). In patients with Alzheimers disease a decrease in high-affinity
nicotine binding sites is one among other changes in the brain (Nordberg and
Winblad 1986; Whitehouse et al 1986). Parkinsons disease is also associated with a
large loss of high-affinity nicotine binding sites in the brain (Perry et al 1995).
The non-neuronal cholinergic systemGeneral
The term non-neuronal cholinergic system is based on the fact that ACh is found in
cells other than neurones. Dale (1914) and Ewins (1914) obtained the first evidence
of the presence of ACh in plants.
The occurrence of acetylcholine has been analysed in human placenta (Rowell and
Sastry 1981), bronchial epithelial cells (Klapproth et al 1997; Wessler et al 1995)
mononuclear cells (Fujii et al 1996), sperm (Sastry and Sadavongvivad 1978), retina
(Hutchins and Hollyfield 1986) and keratinocytes (Grando et al 1993b; Klapproth et
al 1997).
A variety of non-neuronal tissues synthesise and degrade ACh. Expression of the
ChAT protein has been found in non-neuronal cells such as bronchial epithelial cells
(Klapproth et al 1997; Reinheimer et al 1996), keratinocytes (Grando et al 1993b),
-
7/30/2019 Patogenesis Ppp
18/56
18
cells of the human small and large intestine (Klapproth et al 1997) and placental cells
(Rowell and Sastry 1981).
AChE has also been found in the placenta (Rowell and Sastry 1981) and in
heamatopoietic cells, i.e. red blood cells (Herz and Kaplan 1973), platelets and T
lymphocytes, but not B lymphocytes (Szelenyi et al 1982). In a colonic biopsy
specimen, expression of AChE mRNA has been detected in mast cells with high-
affinity receptors for IgE (Nechushtan et al 1996).
Furthermore, some of these cells have been shown to express ACh receptors.
Macklin et al (1998) have reported that human vascular endothelial cells express the
subunits that form functional nAChR similar to the nAChR expressed by ganglionic
neurones ( -3, -5, -2 and-4). These subunits have also been detected in human
bronchial epithelial cells (Maus et al 1998), where Zia et al (1997) found the-3, -
4, -5 and -7 subunits. The alpha-7 subunit of nAChR is also expressed by
endothelial cells (Conti-Fine et al 2000), and the -3 and -4 subunits have been
found on lymphocytes (Hiemke et al 1996).
The muscarinic receptor subtypes m2 and m3 have been found in human
mononuclear leucocytes (MNLs) (Bronzetti et al 1996). Hellstrm-Lindahl and
Nordberg (1996) found the mRNAs for the m3, m4 and m5 muscarinic subtypes in
MNLs and also in purified T cells. Fujino et al (1997) observed expression of the m1
and m2 subtypes in human lymphocytes. Human skin fibroblasts have been reported
to express m2, m4 and m5 mAChR subtypes (Buchli et al 1999).
The non-neuronal cholinergic system in the skin
Grando et al have made detailed investigations of the non-neuronal cholinergic
system in the skin. They demonstrated that human keratinocytes express the ChAT
protein and also synthesise and secrete ACh (Grando et al 1993b). Furthermore, they
showed that the human epidermis expresses AChE and also nicotinic and muscarinic
AChR (Grando 1997; Grando et al 1995a). The ion channels on keratinocytes are
similar to those expressed by ganglionic neurons, since the-3, -5, -2 and-4
subunits, which are found on keratinocytes, are known to form functional receptors in
several combinations among themselves (e.g.32, 325, 34, 345 or
3245) on ganglionic neurons, and the-7 subunit, which is also present on
keratinocytes, can form functional nAChRs of its own (Grando et al 1995a).
-
7/30/2019 Patogenesis Ppp
19/56
19
Keratinocyte ACh, like neuronal ACh, uses Ca2+ as a second messenger (Grando et al
1996). Ion fluxes mediated by nAChR channels are essential for maintaining
keratinocyte viability, as demonstrated in experiments using muscarinic and nicotinic
blocking agents, where interruption of nicotinic, but not muscarinic, pathways of ACh
signalling was found to inhibit keratinocyte division and result in premature cell death
(Grando et al 1993a). Both nicotinic and muscarinic AChRs regulate cell adhesion
and motility, since blocking by -bungarotoxin and mecamylamine, specific AChR
antagonists, caused cell detachment and abolished cell migration (Grando et al
1995a).
High affinity mAChRs have been found on keratinocyte cell surfaces in a high
density (Grando et al 1995b). These receptors mediate effects of muscarinic drugs on
keratinocyte viability, proliferation, adhesion, lateral migration and differentiation.
The mAChR subtypes m1, m3, m4 and m5 have been found in the epidermis (Ndoye
et al 1998). Keratinocytes expressed a unique combination of mAChR subtypes at
each step of their development in the epidermis and it was proposed that each
receptor may regulate a specific cell function (Ndoye et al 1998).
Effects of acetylcholine on cells
Non-neuronal ACh can exert its effects through different pathways. Acetylcholine
released from non-neuronal cells activates the membrane-bound AChR (nicotinic and
muscarinic) localised on the same (autocrine effects) or on neighbouring cells
(paracrine effects). Klapproth et al (1997) reported that the mitogenic effect of ACh
on cultured human bronchial epithelial cells was counteracted by antagonists of
nicotinic and muscarinic receptors. Thus, the effect of ACh is mediated by the
classical extracellular membrane-bound receptors. Interestingly, in the same study it
was found that the ChAT blocker, bromoacetylcholine, had a stronger antiproliferative effect than a combination of the two antagonists blocking the
nicotinic and muscarinic receptors respectively, which may suggest that ACh also has
cytosolic action.
Experimental evidence has been presented that ACh is involved in the regulation of
the mitotic cycle of epithelial cells in humans (Cavanagh and Colley 1989; Grando et
al 1995a; Grando et al 1993b; Klapproth et al 1997). Furthermore, detection of ACh
and its receptors in immune cells indicates that it may take part in the immune system
-
7/30/2019 Patogenesis Ppp
20/56
20
by activation and proliferation of these cells [reviewed in Kawashima and Fujii
(2000)].
Nicotinic influence on the neuronal chol inergi c system
Nicotine acts agonistically on nAChR, and is thus able to reproduce the same effects
as ACh on cells expressing these receptors, but in contrast to ACh, nicotine is not
degraded by AChE. The nAChR channel opens in response to the binding of agonist
(activation) but also becomes refractory to activation during prolonged exposure to
nicotinic agonists (desensitisation) (Peng et al 1994). The nicotine concentrations
required to desensitise the receptor are nearly 1000 times lower than those required
for activation [reviewed by Changeux (1990)]. It has been reported that smoking
increases the number of nAChRs in the human brain (Breese et al 1997). It is
proposed that nicotine-induced up-regulation of neuronal nicotinic receptors results
from a decrease in the rate of receptor turnover (Peng et al 1994). The increase may
also be due to an adaptive response of neurones to accumulation of chronically
desensitised receptors. Alpha-3 nAChRs are more resistant to desensitisation than
other receptor subtypes, such as those containing the -4 and -7 subunits (Olale et al
1997).
on the non-n eur onal choli nergic system
Nicotine is present in high concentrations in the blood of smokers (Russell et al 1980)
and might contribute to desensitisation of the nAChRs and in this way influence their
normal function. There are reports that smoking increases the number of nAChRs on
human bronchial epithelial cellsin vivoand that nicotine increases the number of
nAChRsin vitroon these cells (Zia et al 1997), and that long-term exposure of
respiratory epithelial cells to nicotine increases their Ca2+ concentration, which could
lead to cell damage. Smoking also increased the number of nAChRs on
polymorphonuclear cellsin vivo(Benhammou et al 2000; Lebargy et al 1996)
Lebargy et al (1996) reported that the increase in binding sites persisted for several
months and did not return to the non-smoking level until one year after cessation of
smoking.
-
7/30/2019 Patogenesis Ppp
21/56
21
Alpha-7 nAChRs have been found in small cell lung carcinomas, and the growth of
cell lines derived from these tumours was inhibited by an -7-specific antagonist
(Chini et al 1992; Codignola et al 1996; Quik et al 1994). Thus, in small cell lung
carcinomas in smokers the cancer growth may be facilitated by nicotine stimulationof -7 receptors.
on keratinocytes
Short-term exposure to nicotine stimulates cytoplasm motility and lateral migration of
cultured keratinocytes (Grando et al 1995a). Other (keratinocyte) functions such as
proliferation, adhesion and differentiation may also be affected as a result of
accelerated ion exchange through nicotinic channels. Chronic exposure to nicotineabolishes migration of cultured human keratinocytes in a dose-dependent manner
(Lee et al 1996). Furthermore, chronic nicotine exposure leads to an increase in the
number of keratinocytes forming cornifying envelopes, as well as in the expression of
filaggrin, involucrin and transglutaminase type 1 (Grando et al 1996). Zia el al (2000)
found that keratinocytes incubated with nicotinein vitroexpressed a higher
percentage of the -7 nAChR.
De Hertog et al (2001) concluded that tobacco smoking is probably a risk factor for
cutaneous squamous cell carcinoma. Current smokers were found to be at higher risk
than former smokers, and a clear relation to the number of cigarettes currently
smoked was also observed.
on endotheli al cell s
Nicotine is known to induce vasoconstriction. The cutaneous blood flow, as measured
with a laser Doppler flowmeter, was decreased both in habitual smokers and in non-
smokers after smoking a single cigarette (Monfrecola et al 1998). The micro-
circulation showed a slower recovery phase in the smokers, however, indicating
adaptation to smoke.
There are functional nicotinic receptors on the endothelial cells (Macklin et al
1998). Since in tobacco users the concentration of nicotine in the blood is high
(Russell et al 1980), these receptors may become desensitised after prolonged
exposure to nicotine, which can make them unable to respond in a normal way to the
endogenous ACh.
-
7/30/2019 Patogenesis Ppp
22/56
22
AIMS OF THE INVESTIGATION
The general aim of this investigation was to study the pathogenesis of palmoplantar
pustulosis.
The specific aims were:
- to define the cellular components of the inflammation in PPP.
- to localise the site of inflammation with particular reference to the eccrine gland
and duct.
- to study the distribution of the general nerve marker PGP 9.5 and of the
neuropeptides substance P and calcitonin gene-related peptide in PPP skin.
- to study contacts between sensory nerve fibres and mast cells in PPP skin.
- to study the distributions of choline acetyltransferase and acetylcholinesterase in
palmar skin from healthy non-smokers and smokers and from PPP patients.
- to study the distributions of the -3 and -7 subunits of the nicotinic
acetylcholine receptor in palmar skin from healthy non-smokers and smokers, and
from PPP patients.
- in view of the association between PPP and autoimmune diseases, to address the
question of whether PPP itself might be an autoimmune disease by measuring
serum antibodies to nAChR and by screening PPP sera for immunofluorescence
in healthy palmar skin.
-
7/30/2019 Patogenesis Ppp
23/56
23
PATIENTS AND METHODS
PatientsPaper I, II, III and IV
Anamnestic data
Fifty-nine patients (52 women, 21-79 years old, and 7 men, 43-71 years old) with
typical PPP of the palms and/or soles answered a questionnaire. Their smoking habits
over the years was also investigated.
Clinical examination
Thirty-nine of the patients (35 women, 4 men) who answered the questionnaire were
examined clinically (GM). The degree of erythema and scaling was graded from 0 to
4 and the number of fresh pustules was counted. The patients were only using
emollients at the time of the examination. None of the patients were taking beta-
blockers or lithium.
Blood samples
Sera from the patients were analysed by routine methods for: triiodothyronine,
thyroxine, thyroid stimulating hormone, immunoglobulins (IgG, IgA, IgM and IgE),
eosinophilic cationic protein and antibodies (ab) to thyroglobulin, thyroid peroxidase,
parietal cells and gliadin (IgA and IgG).
BiopsiesAfter intradermal injection of xylocaine-adrenaline, one to three 3-mm punch biopsy
specimens were taken from involved skin and in some patients also one from
seemingly non-involved skin.
The specimens were either fixed in buffered 4% formalin and embedded in paraffin,
or snap-frozen in 70oC, or fixed in 4% paraformaldehyde with 0.2% picric acid
(Lanas fixative) for one hour and then rinsed in 0.1 M Srensens buffer containing
10% sucrose for at least 24 h before they were frozen.
Paper V
In this study sera from the 39 patients described above and six new sera were used
(39 women, 19-71 years old; 6 men, 36-70 years old). At the onset of PPP 43 patients
were smokers. At the time of the present study 9 had stopped smoking or had reduced
the number of cigarettes in recent years.
-
7/30/2019 Patogenesis Ppp
24/56
24
Reference persons
The reference group consisted of two subgroups: smokers and non-smokers. All of
the smokers had been smoking for many years.
The number of smokers and non-smokers varied in the different studies:
Papers I and II: 2 smokers (1 woman, 1 man) and 7 non-smokers (6 women, 1 man),
all of them healthy.
Papers III and IV:7 smokers (5 women, 2 men) and 8 non-smokers (7 women, 1
man), all of them healthy.
Three punch biopsy specimens, which were handled in the same way as the
specimens from the patients, were taken from palmar skin in all of these persons.
Paper V: In this study serum samples were taken from 23 patients with palmar
eczema. Of these patients, 15 had smoked for many years, but six of them had
stopped smoking in recent years.
One 3-mm skin punch biopsy specimen was taken from healthy non-smoking and
smoking persons, from the hypothenar region after intradermal injection of xylocaine-
adrenaline. For comparison, biopsy specimens were also taken from the dorsal aspect
of the forearm and from the gluteal region. These specimens were snap-frozen at
-70oC.
Immunohistochemistry Peroxidase and alkaline phosphatase methods
Detailed descriptions of the different methods used in these studies are given in the
respective papers.
Table 1a presents an overview of the different antibodies used, the fixatives, and thestaining techniques employed.
In all specimens endogenous peroxidase activity was blocked by incubation in 0.3%
H2O2 in phosphate buffered saline (PBS) for 15 min. Between the incubations the
sections were rinsed in PBS twice for 5 min.
Controls with IgG of the same isotype and in the same dilution as the primary
monoclonal antibodies were negative. Polyclonal antibodies gave no staining when
they were preabsorbed with their corresponding peptides.
-
7/30/2019 Patogenesis Ppp
25/56
25
T a b
l e 1 a
. A n t
i b o d
i e s u s e d
i n p e r o x i
d a s e a n
d a l
k a l i n e p h o s p h a t a s e m e t
h o d s
.
P a p e r
n u m
b e r
A n
t i g e n
A n t i b o d y
V i s u a l
i z i n g
D i l u t i o n
S o u r c e
F
i x a t
i v e
T e c
h n
i q u e
I
E o s
i n o p
h i l c a t
i o n i c p r o t e i n
A n t i - E G 2
E o s
i n o p
h i l s
1 / 2 0 0
K a b
i P h a r m a c
i a
A
c e t o n e
P A P
A P A A P *
I
H u m a n n e u t r o p h
i l l i p o c a l
i n
( H N L ) ( S e v e u s e t a l
1 9 9 7 )
A n t i - H N L
N e u
t r o p
h i l s
1 0 u g / m
L
P h a r m a c
i a D i a g n o s
t i c s
M
e t h a n o
l
A P A A P *
I
C D 3
A n t i - C D 3
L y m p h o c y t e s
1 / 1 0 0
B e c
t o n -
D i c k i n s s o n
A
c e t o n e
P A P
I
T r y p
t a s e
M A B 1 2 2 2
M a s
t c e l
l s
1 / 5 , 0 0 0
C h e m
i c o n
I n t . I n c .
F o r m a l
i n
P A P
I
K e r a t
i n s
( W a t a n a b e e t a l
1 9 9 3 )
A E 1 / A E 3
S w e a
t g l a n d
a p p a r a
t u s
1 / 1 , 0 0 0
B o e
h r i n g e r M a n n h e i m
C o r p .
F o r m a l
i n
P A P
I I I
C h A T
A n t i - C h A T
C h A T
1 / 5
B o e
h r i n g e r M a n n h e i m
C o r p .
L a n a
A B C * *
I I I
C h A T
M A B 3 0 5
C h A T
1 / 2 5 0
C h e m
i c o n
I n t . I n c .
A
c e t o n e
A B C
I I I
A C h E
M A B 3 0 3
A C h E
1 / 6 0 0
C h e m
i c o n
I n t . I n c .
A
c e t o n e
A B C
I V
- 3 n A
C h R s u
b u n i
t
m A B 3 1 3
n A C h R
1 / 3 , 0 0 0
R B I
L a n a
A B C * *
I V
- 7 n A
C h R s u
b u n i
t
A C h R - 7
n A C h R
1 / 5 0
S a n t a
C r u z
I n c .
A
c e t o n e
A B C
* T h e a l k a
l i n e p h o s p h a t a s e a n t
i a l k a l
i n e p h o s p h a t a s e
( A P A A P ) t e c h n i q u e w a s u s e d , a
s n e u t r o p
h i l s c o n t a i n p e r o x i
d a s e , w
h i c h c o u l
d g i v e
f a l s e p o s i
t i v e s t a i n i n g .
* * S e c
t i o n s u s e d w e r e
1 4 u m , i
n o t
h e r s
t a i n i n g s
6 u m s e c t
i o n s w e r e u s e d .
T h e n u m
b e r o
f s t a i n e d c e
l l s w a s c o u n
t e d i n t h e e p
i d e r m
i s , p
a p i l l a r y
d e r m
i s ( b e l o w
t h e p u s t u l e w
h e n a p p l
i c a b
l e ) a n d
i n t h e r e
t i c u l a r d e r m
i s .
T h e s e c t
i o n s s t a i n e
d
w
i t h A E 1 / A E 3 w e r e
t r e a
t e d w
i t h 0 . 0 5 % p r o t e a s e f o r
1 0 m
i n b e f o r e s t a i n i n g . A
E 1 / A E 3 c o n
t a i n s a n
t i b o d
i e s a g a i n s
t c y t o k e r a t
i n s
1 - 8 , 1 0
, 1 4 / 1 5
, 1 6 , a n
d 1 9
. S e c
t i o n s
f r o m
t y l o t i c e c z e m a w e r e a l s o s t a i n e
d w
i t h A E 1 / A E 3 f o r c o m p a r i s o n .
T h e A
C h R - 7 a n
t i b o d y w a s
t h e o n
l y o n e
t h a t w a s p o
l y c l o n a l .
-
7/30/2019 Patogenesis Ppp
26/56
26
To reduce the possible variations in staining intensities, all specimens used for one
antibody were stained on the same day. All immunohistochemical and
immunofluorescence evaluations were made on coded slides.
In the vital epidermis the staining intensity was estimated in the different strata as
follows: unstained = 0, weak = 1, medium = 2 and strong = 3. The numbers of
unstained and of ChAT-, AChE-,-3- and -7-positive ducts in the reticular dermis,
in the papillary dermis and in the vital epidermis and coils were counted. All visible
dermal ducts were counted as one duct each. The proportions of weakly and strongly
stained coils and ducts in the reticular dermis were calculated by dividing their
number by the total number (unstained and stained) of coils and ducts in the reticular
dermis. The numbers of immunoreactive cells in the papillary dermis and reticular
dermis and below the pustules were counted and classified as very few (0-3), few (4-
10) or many (>10).
One immunohistochemical double staining was performed (Table 1b).
Table 1b. Double staining with ABC and APAAP techniques.
Paper number Antigens Visualising Techniques
III AChE and chymase1 AChE in mast cells ABC APAAP
1The chymase antibody was used to verify that only mast cells were AChE+, since this antibodyworked better than the tryptase antibody in this double staining. Both antibodies were monoclonal.Sections 6m thick were used.
Immunofluorescence
Table 2a shows the different antibodies used, the fixatives, and the staining
techniques employed. Five non-adjacent sections were placed on each slide.
Table 2a. Antibodies used in immunofluorescence stainings.
Papernumber
Antigen Antibody Visualising Dilution Source Fixative Secondaryantibodyconjugated with
II PGP 9.5 Anti-PGP 9.5 Nerve fibres 1/800 Biogenesis Lana TRITCII Substance P Anti-SP Substance P 1/400 Peninsula Lana TRITCII Calcitonin gene-
related peptideAnti-CGRP CGRP 1/400 Peninsula Lana TRITC
PGP 9.5 = protein gene product 9.5; TRITC = Tetramethyl-rhodamine-isothiocyanate; All antibodies were
polyclonal and 14m thick sections were used.
-
7/30/2019 Patogenesis Ppp
27/56
27
Six compartments in all specimens were analysed: the epidermis, dermo-epidermal
junction, papillary dermis, reticular dermis, eccrine sweat glands and their ducts and,
wherever applicable, beneath pustules. Each separate fragment of nerve fibre was
considered as one fibre. All five sections were analysed and the mean values per
square millimetre or millimetre of epidermal length were calculated. Image analysis
of the nerve fibres around the sweat glands was performed. The area of the positive
nerves was expressed in per cent of the total sweat gland area.
Three immunofluorescence double stainings were performed (Table 2b).
Table 2b. Double stainings with immunofluorescence techniques.
PGP 9.5 = Protein gene product 9.5; HNL = human neutrophil lipocalinFITC = Fluorescein isothiocyanate; TRITC = Tetramethyl-rhodamine-isothiocyanateSections were 14m thick.*All close contacts between mast cells and nerve fibres in the papillary dermis were counted and thenumber of such contacts per mm of epidermal length was calculated.
Serum immunofluorescence (paper V)
Sections from palmar skin of healthy controls (non-smokers and smokers), 6 m thick
and fixed in acetone, were incubated overnight at +4C with serum (dilution 1/150)
from 45 patients with PPP and 23 patients with palmar eczema. Immunofluorescence
stainings were also performed with sera from 7 patients with myasthenia gravis, all of
whom had elevated serum concentrations of nAChR antibodies. Fluorescein-
isothiocyanate (FITC)-anti-human IgG (dilution 1/40; Dakopatts, Glostrup, Denmark)was used as secondary antibody. Control with FITC anti-human IgG omitting the
patient serum was negative. All parts of the sweat gland apparatus (duct and gland),
epidermis and dermis were studied for the presence of staining. The staining intensity
was classified as weak (+), medium (++) or strong (+++).
Double staining: endothelium palmoplantar pustulosis serum (paper V)
This double staining was performed to confirm that the immunofluorescence obtained
with the PPP sera was localised on endothelial cells. Sections 6 m thick and fixed in
Papernumber
Antibodies against Visualizing Secondary antibodyconjugated with
II Tryptase and PGP 9.5 Contacts mast cells - nerve fibres* Texas Red - FITCII Substance P and HNL Neutrophils containing substance P TRITC - FITCII Substance P and EG2 Eosinophils containing substance P TRITC - FITC
-
7/30/2019 Patogenesis Ppp
28/56
28
acetone were incubated with two mouse monoclonal anti-human endothelial
antibodies, Q bend 10 (dilution 1/40; Skybio, Bedfordshire, UK) and CD 31 (dilution
1/40; Dakopatts), overnight at 4C. Biotinylated horse antimouse IgG (dilution 1/200;
Vector, CA, USA) was used as secondary antibody. Subsequently the sections were
incubated with Texas Red Streptavidin (dilution 1/100; Vector) for 30 min and then
with 10% normal mouse serum (Dakopatts) for 60 min. The sections were then
allowed to react with 10% normal rabbit serum for 10 min and thereafter with serum
from the patients and FITC-anti-human IgG as above. To rule out non-specific
staining, including overlapping between the fluorescence filters, three control
stainings were performed: one using mouse IgG of the same isotypes and dilutions as
the primary endothelial antibodies plus patient serum, another with mouse IgG and
omitting the patient serum, and as a third control the endothelial antibodies were used
without the patient sera.
Western blot (paper III)
Western blot analysis was run on cell extracts from pure preparations of neutrophils
and eosinophils from peripheral blood from healthy donors. Granulocytes from a
Ficoll preparation were incubated with supermagnetic particles coupled to a
monoclonal antibody against CD 16, a molecule present on neutrophils but not on
eosinophils (Hansel et al 1991). The cell preparations were kindly provided by
Associate Professor Lena Hkansson, Section of Clinical Chemistry, Department of
Medical Sciences, University Hospital, Uppsala. Protein extract from human placenta
was used as a positive control (Rowell and Sastry 1981).
The proteins were separated on 10% SDS-PAGE ready-gels (Bio Rad, CA, USA),
and blotted on a nitrocellulose membrane. ChAT was visualised with a polyclonal
rabbit-anti-ChAT antibody (dilution 1/1,000; Biogenesis, Poole, UK) and an Immun-Blot kit with an alkaline phosphatase conjugate (dilution 1/3,000; Bio Rad). As a
negative control, placenta extract was used as above, but without the primary
antibody.
Radioimmunoassay (paper V)
Serum nAChR antibodies were measured in sera from 45 PPP patients and, for
comparison, in sera from 10 patients with palmar eczema, with a radioimmunoassayused for determination of acetylcholine receptor antibodies in myasthenia gravis
-
7/30/2019 Patogenesis Ppp
29/56
29
(Lefvert et al 1978). In brief, a preparation of cholinergic receptors from human
skeletal muscle was incubated with radiolabelled alpha-bungarotoxin, serum was
added and the toxin-receptor-IgG complex was precipitated using anti-human IgG.
The precipitate was separated and washed by centrifugation. Radioactivity (CPM)
was determined and the concentration of receptor antibodies in arbitrary units was
calculated.
StatisticsThe statistical significance of differences was calculated by the Mann-WhitneyU -test
(papers I, II, III, IV and V) or Fishers exact test (paper V).
-
7/30/2019 Patogenesis Ppp
30/56
30
RESULTS AND DISCUSSION
Paper I Anamnestic data
The worsening effect of warm weather and stress in a high proportion of patients
indicated that the sweat gland apparatus might be a possible target for the
inflammation. The fact that 95% of the patients were smokers at the onset of the
disease (at a mean age of 42 years, range 15-66 years) pointed to nicotine as a possible
precipitating factor for the disease (Table 3).
Table 3. Anamnestic data in 59 patients (52 women, 7 men) with palmoplantar pustulosis.
Per centHeredity for -palmoplantar pustulosis 14-psoriasis 22-thyroid disease 22-gluten intolerance 3
Patients with history of -psoriasis 10-thyroid disease 14-gluten intolerance 8-diabetes 7-vitiligo 5-alopecia areata 3
Stress preceding onset of PPP 25Smoker at onset of PPP 95Worsening associated with- hot weather 36- stress 46
Pruritus 95Arthralgia 42
There was a high prevalence of a number of autoimmune diseases among the PPP
patients, as shown in Table 3.
The association between autoimmune thyroid disease and PPP is well known and has
been investigated in detail by Rosn (1988). The majority of the PPP patients with
-
7/30/2019 Patogenesis Ppp
31/56
31
thyroid disease had hypothyroidism, the prevalence of which in Swedish women is 1.9
percent (Hallengren 1998).
The increased prevalence of coeliac disease in PPP patients has not been reported
previously. The prevalence figures for coeliac disease in the Swedish population have
increased in the last few years since the introduction of screening for antibodies to
gliadin and endomysium and recently also to tissue transglutaminase. Silent coeliac
disease has been diagnosed in 0.3% of Swedish blood donors (Grodzinsky 1996).
Recent data from a screening study of children in Northern Sweden indicate a
prevalence of at least 1% (Carlsson et al 2001). Since January 2001 anamnestic data
have been available for 82 patients with PPP. The prevalence of previously diagnosed
coeliac disease in this extended group is 6%. One of our patients who was found to
have coeliac disease had disabling PPP, but since the introduction of a gluten-free diet,
her PPP has totally cleared, indicating that gluten intolerance might be of pathogenetic
relevance in PPP.
There was also a high prevalence of diabetes among the PPP patients. This has
become even more evident since the number of patients has increased (to 82)
compared with the number at the start of the study. Twelve of the 82 patients (14.6 %)
had diabetes; 9/12 were < 50 years old. At screening for diabetes in the community of
Lax, Sweden, 0.8% of the screened women aged 25-44 years and 2% of those aged
45-54 years were found to have diabetes (type 1 or type 2) (Andersson et al 1991).
Four of the patients (4.8%) had type 1 diabetes. The prevalence of type 1 diabetes
among women in the community of Lax was 0.3-0.4%. Thus there is a marked
increase in the prevalence of diabetes in PPP, which has not been reported previously.
However, a predisposition to diabetes in PPP was discussed in a Japanese study, as a
diabetic pattern at an oral glucose tolerance test was found in 22% of the patients
(Uehara 1983), but there are no other reports on such an association. There are,however, previous reports of an increased prevalence of type 2 diabetes in psoriasis
(Binazzi et al 1975). A significantly raised prevalence of psoriasis and vitiligo has
been reported in children with type 1 diabetes (Montagnani et al 1985).
The high prevalence of associated autoimmune disease in PPP gave us reason to
consider the possibility that PPP itself might be an autoimmune disease affecting the
skin and also the joints.
-
7/30/2019 Patogenesis Ppp
32/56
32
Clinical findings
Erythema and scaling were present in all but one patient. Fresh pustules were observed
in 26 patients (range 1-100). Patients with the highest cigarette consumption had the
largest mean number of pustules (but there were large variations and the groups of
patients were small).
The association between PPP and autoimmune disease was further strengthened by
the presence of antibodies to thyroglobulin/thyroperoxidase in 25% of the patients.
IgA antibodies to gliadin were present in 25%, compared to 9% in female healthy
blood donors (Lindquist et al, unpublished data). Among patients with psoriasis
vulgaris 16% had IgA antibodies to gliadin (Michalsson et al 1993), thus the
prevalence of IgA antibodies to gliadin is even higher in PPP.
The significantly elevated mean serum IgA and decreased IgM are similar to the
pattern present in coeliac disease and dermatitis herpetiformis (O'Mahony et al 1990)
and also in psoriasis (Michalsson et al 1995) and psoriatic arthritis (Lindqvist el al,
unpublished data), indicating that the intestinal mucosa might also be involved in PPP.
Elevated serum ECP in PPP was previously reported by Lundin et al (1990) and
suggested that the eosinophil granulocyte was activated. The results of the present
study further confirm that eosinophil granulocytes are involved in the inflammation
(see below).
Inflammatory cells
The pustules were found to contain large numbers of eosinophils, an observation not
made previously, as well as neutrophils, indicating that the eosinophils participate in
the pustule formation together with neutrophils. Furthermore, numerous eosinophils
were present in the papillary dermis below the pustules. Another previously
unreported feature was the massive infiltrates of mast cells in the upper dermis,especially in specimens with pustules. One important chemoattractant for both
neutrophils (Baggiolini et al 1989) and activated eosinophils (Burrows et al 1991) is
IL-8, which has been shown to be present in the eccrine duct in general and in the
epidermis in PPP skin (Anttila et al 1992; Jones et al 1995). Furthermore, mast cells
have been found to secrete IL-8 (Ansel et al 1997).
There was a massive infiltrate of lymphocytes in the papillary dermis, with a
tendency to accumulation below the pustule. The accumulation of the participating
-
7/30/2019 Patogenesis Ppp
33/56
33
inflammatory cells below the pustules may indicate that there is an epidermal target
for the inflammation that is not evenly distributed in the epidermis.
Fig. 3. Schematic drawing of the participating inflammatory cells in PPP.
The sweat gland and duct In the specimens from involved PPP skin no acrosyringia were visible with the
keratin antibody AE1/AE3, reported to give staining in the sweat gland apparatus
(Watanabe et al 1993), in contrast to the findings of acrosyringia in the control
specimens, indicating that the intraepithelial duct may be destroyed in PPP, which
might reflect an inflammatory process at this site. This feature may have pathogenetic
relevance in PPP, since there were no changes in the appearance of the acrosyringia
in specimens from our tylotic eczema patients or in those from patients withdyshidrotic eczema (Kutzner et al 1986). We also stained the sweat pores in the
palmar skin of PPP patients with the iodine-starch method and compared the pattern
with that in the palms of healthy persons (no data shown). In the PPP patients a
diffuse pattern was observed, whereas in the control persons there was a distinct
pattern of black spots in even rows over the entire palm.
-
7/30/2019 Patogenesis Ppp
34/56
34
Paper II PGP 9.5, substance P and calcitonin gene-related peptide
In view of the seemingly strong influence of stress on the inflammatory activity in
PPP and the itching when new pustules were formed, a study of the distribution of
nerve fibres and the presence of SP- and CGRP-like immunoreactivity (-LI) in
involved skin in PPP patients and in healthy controls was undertaken.
The innervation of the sweat glands was studied with a general nerve marker,
protein gene product 9.5 (PGP 9.5). In the patients the nerve fibres around the sweat
glands seemed to be more or less fragmented, while in the controls they encircled the
sweat gland without any interruptions (Fig. 4a and b). Image analysis showed that
there were significantly fewer fibres around the sweat glands in the patients than in
the controls (p=0.0006).
Fig. 4. PGP 9.5-positive nerve fibres around a sweat gland in involved palmar PPP skin (a) (note thefragmented fibres) and in palmar control skin (b) (x300).
The largest numbers of nerve fibres with SP- and CGRP-LI were observed in the
papillary dermis, both in patients and controls. There was a tendency to a larger number of nerve fibres with SP- and CGRP-LI in the patients, especially in the
papillary dermis. In the reticular dermis nerve fibres with SP- and CGRP-LI were
localised close to blood vessels and some were also observed close to the sweat ducts.
Whether the probable damage to the nerves around the sweat gland is a result of the
inflammation higher up in the papillary dermis is not known. Nor is it known whether
this has an influence on the function of the sweat gland. One possibility is that the
damage may be induced by neurotoxic effects of eosinophil granule proteins, as large
-
7/30/2019 Patogenesis Ppp
35/56
35
numbers of eosinophils have been found in the pustules and also in the upper
papillary dermis (Eriksson et al 1998).
Both psoriasis and PPP patients experience worsening of their skin disease during
periods of stress. An increased number of CGRP-positive nerve fibres has been
observed in the papillary dermis of psoriasis patients with high stress levels (Harvima
et al 1993). CGRP induces vasodilation, resulting in long-lasting erythema
(Wallengren and Hkanson 1987). An increased number of SP-positive nerve fibres
(Naukkarinen et al 1989) and an increased number of contacts between SP-containing
nerve fibres and mast cells (Naukkarinen et al 1996) have also been found in psoriatic
lesions, indicating neurogenic involvement in the inflammation in psoriasis.
There are discrepancies in the numbers of nerve fibres reported from different
studies, which may indicate that there are difficulties in counting nerve fibres, and the
results may depend on the methods employed. In this study all nerve fibres were
counted, including nerve fragments, and the number varied considerably, both in
controls and patients. With image analyses, where the number of immunoreactive
nerve fibres could have been related to the area of the epidermis, different results
might have been obtained, since the epidermis in PPP skin is much thicker than in
control palmar skin. There are similar problems in comparing the number of nerve
fibres in the papillary dermis with that in healthy skin.
Nerve fibres and their contacts with mast cells
Close contacts between nerve fibres and mast cells provide an opportunity for
neuropeptides to degranulate these cells, and release of histamine and several other
degranulation products will further enhance the inflammatory reaction. The itching
that occurred when new PPP pustules were formed might be explained by histamine
release caused by neuropeptides.The number of tryptase-positive mast cells in the papillary dermis was larger
(p=0.0003) in the lesional palmar skin from PPP patients than in the healthy controls,
and the number of contacts between mast cells and nerve fibres with PGP 9.5-LI was
also significantly larger (p=0.02).
As the number of nerve fibres was similar in PPP and control skin, but the number
of mast cells was three times larger in the PPP skin than in the controls, the increased
number of contacts may be due to the increase in mast cells.
-
7/30/2019 Patogenesis Ppp
36/56
36
It has been reported (Naukkarinen et al 1996) that both the number of mast cells and
the number of SP-positive nerve fibres in contact with mast cells are increased in
psoriatic lesions. The increased number of contacts in PPP and in psoriatic skin may
imply more pronounced neurogenic involvement in the inflammation than in
conditions without a mast cell increase.
Neuropeptide immunoreactivity in granulocytes
PGP 9.5- and SP-LI, but not CGRP-LI, were present in granulocytes. With use of
double staining, neutrophils showed SP-LI, whereas eosinophils were SP-negative.
The neutrophils were situated in the pustule, or were visible in the papillary dermis,
and were observed within the sweat duct in the papillary dermis (possibly migrating
towards the pustule) (Fig. 5).
Fig. 5. SP-positive granulocytes (neutrophils) in the sweat duct, possibly migrating towards the
pustule, and in the pustule (x475).
Substance P has previously been detected in human peripheral leucocytes from
healthy subjects (De Giorgio et al 1998). SP-LI has also been found in neutrophils in
infiltrates in psoriatic lesions (Pincelli et al 1992). This suggests that neutrophils are
another possible source of SP, both in PPP and in psoriatic skin. As there is a massive
granulocyte infiltration in PPP, the possible influence of SP may be more pronounced
in this disease than in psoriasis, although it is probably of importance in both
-
7/30/2019 Patogenesis Ppp
37/56
37
conditions. SP has been reported to stimulate proliferation of cultured keratinocytes in
a dose-dependent manner (Tanaka et al 1988). Keratinocytes in PPP skin might be
influenced in a way similar to that in psoriasis, where their proliferation rate is
increased.
Paper IIIChAT and AChE in the epidermis and sweat gland apparatus
The mechanisms underlying the
inflammation in the acrosyringium
in PPP are not known. Sweating
and the sweat gland apparatus
seem to play an important role in
the pathogenesis of PPP.
Sympathetic fibres innervate
the sweat glands, but are cholinergic,
and ACh is the main inducer of
sweating (Fig. 6). Fig. 6.
As the ACh-synthesising enzyme,
choline acetyltransferase, and the degrading enzyme, acetylcholinesterase, regulate
the ACh level, the distribution of ChAT- and AChE-LI was studied in normal palmar
skin in non-smokers and smokers and in involved skin in PPP patients.
In addition to observing ChAT- and AChE -LI in the epidermis, we found that the
eccrine glands and ducts displayed more intense ChAT and AChE reactivity than the
epidermis. Table 4 shows some important findings of the ChAT and AChE stainings.
-
7/30/2019 Patogenesis Ppp
38/56
38
Table 4. Results of the ChAT and AChE immunohistochemistry in the epidermis andsweat gland apparatus.
ChAT non-smokers ChAT smokers ChAT involved
PPP
AChE non-smokers,
smokers and involved
PPPStaining intensity/vital epidermis Weak to moderate Weak to moderate Weak to
moderate
Weak
Staining intensity/acrosyringium
in stratum corneum
No staining No staining No staining Strong staining
Number of positive acrosyringia
in vital epidermis and the
presence of strongly stained
acrosyringia
Larger number than in
the smokers and PPP
patients and some were
strongly stained
Larger number than in
the PPP patients and
none were strongly
stained
Smallest number
and none were
strongly stained
No differences between
the groups, almost half of
them were strongly
stained.
Number of strongly stained coils
and reticular ducts
Largest number Medium number Smallest number No differences between
the groups
There were some differences between the distribution of ChAT- and that of AChE-LI.
In the acrosyringium AChE-LI was most intense in the lower part of the stratum
corneum corresponding to the site of the pustule in PPP. This is of special interest,
since there is a homology between AChE and thyroglobulin (Malthiery and Lissitzky
1987), against which many patients have antibodies. The carboxy terminal of thyroglobulin shows up to 64% homology with AChE. (The sites of thyroid hormone
synthesis are clustered at both ends of the thyroglobulin.) No ChAT-LI was present in
the acrosyringium at this level. On the other hand there was marked ChAT-LI in the
acrosyringium in the living part of the epidermis. Klapproth et al (1997) reported that
ChAT enzyme activity and ChAT-LI were to some extent correlated and that high
ChAT enzyme activity and immunoreactivity corresponded to a high ACh content
(skin, intestine) and vice versa (bronchi). Grando et al (1993b) observed ChAT-LIthroughout the epidermis and in epidermal appendages and also found that
keratinocytes have ChAT activity resulting in ACh production. If this is also true for
eccrine sweat glands and ducts, ACh will be produced in both the coil and duct until
it reaches the horny layer.
In the epidermis Grando et al (1993b) found AChE reactivity mainly in the basal
layer (keratinocytes and melanocytes). This was also observed in our specimens,
although the AChE reactivity was much stronger in the eccrine sweat apparatus than
in the interappendageal epidermis.
-
7/30/2019 Patogenesis Ppp
39/56
39
ChAT and AChE in inflammatory cells
Granulocytes, which are abundant in the pustules and the papillary dermis in involved
PPP skin, showed strong ChAT-LI. Western blot analysis of proteins extracted from
purified non-stimulated neutrophils and eosinophils confirmed the presence of ChAT-
like proteins in large amounts in neutrophils and in very small amounts in
eosinophils. Placental extract was used as a positive control (Fig. 7).
Fig. 7. Western blot with a polyclonal ChAT antibody; Pla=positive control, placenta (5 g protein), Neu=neutrophils (7 g protein), Eos=eosinophils (8 g protein). Single arrow indicates ChAT 54 kDand double arrows ChAT 69 kD (dimer).
In the skin there are normally few, if any, neutrophils and eosinophils. Thus noconclusions can be drawn from our results as to the extent to which the ChAT
reactivity in granulocytes in the PPP patients might have been influenced by nicotine.
If there is ChAT activity in these cells, this might be of importance not only regarding
the inflammation in PPP but also in other inflammatory diseases, e.g. psoriasis, atopic
dermatitis and asthma, where these cells play a significant role. Further studies will
be undertaken to see whether ChAT in granulocytes can synthesise ACh.
In the healthy non-smokers and in the PPP patients AChE-LI was seen in 25% of the mast cells, while only 10% of the mast cells in the smoking controls showed
AChE reactivity. This preliminary finding might indicate that smoking can influence
the AChE activity. There are no reports on AChE+ mast cells in the skin and only one
report on the presence of AChE in human mast cells, namely in a colonic biopsy
specimen where AChE mRNA expression was detected in FcRI-positive cells
(Nechushtan et al 1996). There are several reports, however, on interactions between
ACh, histamine and mast cells in inflammatory reactions [reviewed in Masini et al
(1985)].
-
7/30/2019 Patogenesis Ppp
40/56
40
Paper IV
Nicotinic receptors in the epidermis and sweat gland apparatus
A role of nicotine in the pathogenesis of PPP has long been discussed. Several
diseases are thought to be caused or aggravated by smoking, but the mechanisms
underlying this effect are not known. That nicotine plays a role in this respect has
been reported both in psoriasis vulgaris (Plunkett and Marks 1998) and in psoriatic
arthritis (Averns et al 1996). The skin disease with the most obvious association with
smoking is palmoplantar pustulosis. We showed in studies I and II that the target for
the inflammation in PPP is the acrosyringium. Nicotine acts as an agonist on nicotinic
acetylcholine receptors and can influence a variety of cellular functions, like cell
adhesion and motility (Grando et al 1995a) and keratinocyte differentiation (Grando
et al 1996).
In this study we chose to investigate the expression of the two nAChR subunits-3
and -7, as most of the possible variations of nAChRs on keratinocytes would then be
included.
In healthy subjects the epidermis and, to an even greater extent, the eccrine sweat
gland and its duct expressed both the-3 and -7 subunits of the nAChR. It was also
evident that smoking influenced the staining intensity but not the distribution in
the healthy controls.
The strongest epidermal -3 staining was present in the involved PPP skin, where it
was significantly more intense than in the non-smoking controls (Fig. 8a and b).
a. b.
Fig. 8. Schematic drawings of -3 staining in palmar skin from (a) a non-smoking control: 1= weak staining of the acrosyringium, 2= epidermis with weak, even staining, 3= the entrance of the sweatduct in the epidermis results in a thicker epidermis, 4= weak staining of endothelial cells; and (b)involved PPP skin: 1= epidermis with the strongest staining in the upper part of the stratum spinosum,2= the pustule with stained granulocytes, 3=-3-positive granulocytes in the papillary dermis.
-
7/30/2019 Patogenesis Ppp
41/56
41
The strongest staining in the sweat gland apparatus was also seen in the involved
skin. The highest proportion of strongly stained ducts in the reticular dermis in
relation to the area of the sections was again observed in involved PPP skin and this
proportion was significantly larger than in the smoking controls (p=0.01), where the
staining was weakest. Psoriasis specimens were also stained for comparison, and a
pattern similar to that in the PPP specimens was observed (data not shown).
The strongest staining of the -7 subunit in the healthy controls (smokers and non-
smokers) was noted in the keratinocytes in the stratum granulosum, with the most
pronounced intensity in the acrosyringium (Fig. 9a).
In the involved PPP skin, in which the stratum granulosum was abolished, there was
a remarkably different pattern, especially around the acrosyringium and closest to the
pustule, where the surface of the keratinocytes was strongly stained, with a fishnet-
like appearance (Fig. 9b).
a. b.
Fig. 9. Schematic drawings of -7 staining in palmar skin from (a) a non-smoking control: 1= thestrongest staining is seen in the acrosyringium in the stratum granulosum, 2= the strong staining of thestratum granulosum, 3= the entrance of the sweat duct in the epidermis results in a thicker epidermis,4= staining of endothelial cells in the dermis; and (b) involved PPP skin: 1= the fish-net like staining pattern in the epidermis, 2= unstained cells in the pustule, 3= the strong staining of endothelial cells,4= the strongest staining of epidermis is seen closest to the pustule.
The abnormal distribution of the -7 nAChR in the keratinocytes in PPP skin may
affect the differentiation, since keratinocytes are able to synthesise, release and
degrade ACh (Grando et al 1993b). Nicotinic receptors have been found to be
important for the terminal differentiation of keratinocytes. Long-term exposure of
keratinocytes to nicotinein vitrohas been shown to inhibit their migration and to
increase the number of keratinocytes forming cornifying envelopes and also the
expression of filaggrin, involucrin and transglutaminase type 1 (Grando et al 1996).
-
7/30/2019 Patogenesis Ppp
42/56
-
7/30/2019 Patogenesis Ppp
43/56
43
Paper V
Serum antibodies to nicotinic receptors and the immunofluorescence pattern
With regard to the massive inflammatory reaction in PPP skin and the high
prevalence of autoimmune disease, it might be suspected that PPP could be an
expression of one or several autoimmune reactions induced by smoking.
A first step in the testing of this hypothesis was to determine whether antibodies
against nAChR could be detected in PPP serum with the method usually used in
Sweden when diagnosing myasthenia gravis (Lefvert et al 1978).
Increased concentrations of such antibodies, though less increased than in
myasthenia gravis, was found in 19/45 (42%) of the PPP patients. The mean antibody
level in the positive sera was 0.75 arbitrary units/l (range 0.2-3.1). Values below 0.2
were considered normal. Among a Swedish group of patients with myasthenia gravis,
93 % had nAChR ab, with a mean value of 3.60+2.20 (Lefvert et al 1978). None of
the sera from the patients with eczema (non-smokers or smokers) had any nAChR ab,
which rules out the possibility of a non-specific reaction in PPP associated with an
inflammation in palmar skin.
Fig. 10. The antibodies (ab) in PPP sera were mostly found in the patients without thyroid and/or gliadin antibodies, indicating that there are two subgroups of PPP.
Twenty-one of the 45 sera (46.7 %) produced a special fluorescence (IF) pattern on
some cells in the papillary dermis in normal palmar skin from a non-smoker. The IF
staining often formed a chain-like pattern (see cover page).
Of the sera from PPP patients with nAChR antibodies 68% gave this positive IF. On
double staining with a specific anti-human endothelial antibody, the IF was found to
be localised to endothelial cells. Furthermore, in palmar skin from a smoker positive
IF was also observed in the acrosyringium in the upper part of the living epidermis.
nAChR ab no nAChR ab0
20
40
60
80
100
thyroid abthyroid and gliadin ab
gliadin ab
no thyroid or gliadin ab
n=19 n=26
P e r c e n
t
-
7/30/2019 Patogenesis Ppp
44/56
44
Of the sera from PPP patients without nAChR, the same IF pattern was produced by
31%, although mostly with lower intensity (Fig. 11a and b). Sera that had antibodies
for both nAChR and/or thyroperoxidase/thyroglobulin/gliadin gave the strongest IF.
a. b.Fig. 11. (a) More sera from patients with nAChR antibodies (ab) than from patients without suchantibodies gave immunofluorescence staining. (b) The sera with nAChR antibodies produced stainingof stronger intensity than those without such antibodies.
The strong IF staining in the acrosyringium in palmar skin from a smoker illustrates
the important role of nicotine, since the acrosyringium is the main target of the
inflammation. This strengthens our theory of a possible up-regulation of an antigen
by smoking. The strong IF staining with sera from patients with several types of autoantibodies might be due to an overlap between these different antigens. This
might explain the association between smoking and PPP and possibly also between
PPP and autoimmune thyroid disease and coeliac disease.
Only two (8.7 %) of the 23 sera from patients with palmar eczema produced any
positive structures one with weak (non-smoking man) and one with medium
(previously smoking woman) staining intensity - in the papillary dermis.
Sera from 7 patients with myasthenia gravis with high concentrations of nAChR antibodies did not produce any positive IF, indicating that the autoantigen(s) in PPP
and myasthenia gravis are probably not the same.
The results of this study indicate that PPP is an autoimmune disease. The antibodies
that bind to endothelial cells and to the sweat gland duct may play a pathogenetic role
in PPP by activating endothelial cells and enhancing the massive infiltration of
inflammatory cells in the papillary dermis and the migration of granulocytes outwards
into the acrosyringium in the stratum corneum and into the pustules.
nAChR ab no nAChR ab0
20
40
60
80
100stainedunstained
n=19 n=26
P e r c e n
t
nAChR ab no nAChR ab
0
1
2
3
n=19 n=26
S t a i n i n g
i n t e n s
i t y
-
7/30/2019 Patogenesis Ppp
45/56
45
CONCLUSIONS
* The massive infiltrates of lymphocytes